Secreted and transmembrane proteins mediate intercellular communication, driving pathophysiological processes such as inflammation, tumor angiogenesis, and metastasis. Most secreted and many transmembrane proteins are difficult to target with small-molecule drugs, as they lack typical drug binding sites. One approach to this problem is to inhibit secreted and membrane proteins indirectly, by preventing their transport to the cell surface. In this grant application, we describe cotransin, a cyclodepsipeptide that inhibits protein secretion in a substrate-specific manner. We discovered that cotransin blocks one of the earliest steps in the life of a secreted protein: cotranslational translocation into the endoplasmic reticulum (ER), a process that requires a signal sequence at the N-terminus of a nascent polypeptide. By an unknown mechanism, cotransin interferes with the ability of the Sec61 translocation channel to open in response to a minority of signal sequences. The major goal of this project is to design and synthesize new chemical tools that will allow us to dissect the molecular basis of cotransin's biological activity. cotransin